The overall goal of this proposal is to understand the mechanisms by which histone deacetylase (HDAC) inhibitors are neuroprotective. By inhibiting HDACs, the deacetylation of histones is blocked, allowing acetylated regions of the genome to remain transcriptionally active. HDAC inhibitors have the significant advantage over other potential drugs in their ability to promote the transcription of a large cassette of neuroprotectiv genes. By studying them in detail, we can optimize their protective and restorative properties and reduce or eliminate their side effects. To this aim, we are proposing to knock down in vitro each of the Class I HDACs to determine which HDACs are most essential to inhibit to impart neuroprotection in the glutathione depletion model of oxidative stress. Preliminary experiments have shown that HDAC2 knock down is neuroprotective. Thus in subsequent experiments we propose to knock down HDAC2 and any other Class I HDACs, which are protective when knocked down with AAV8 shRNA in a mouse model of ischemic stroke. In this way, we can identify the most important HDACs to inhibit and in the future design more specific inhibitors to target these HDACs. We also aim to improve in vivo testing of HDAC inhibitors in neurological disorders by identifying a biomarker of neuroprotection to determine the optimal dose. As HDAC inhibitors alter the expression of a large number of genes, there are many potential biomarkers that could be used to identify the most neuroprotective dose. Thus we have selected two candidate gene biomarkers that are significantly upregulated (TXNIP and PI4K2b) with a protective dose of Scriptaid. Finally, we will test the HDAC inhibitor, Scriptaid, in a mouse model of ischemic stroke and validate our neuroprotective biomarkers in vivo. Importantly we will test use a negative control compound, Nullscript, which has the same functional groups as Scriptaid but is unable to inhibit HDACs. By using Nullscript, we can rule out many of the potential off-target effects of HDAC inhibitors. With the results from these experiments we stand to significantly expand our knowledge of how HDAC inhibitors, such as Scriptaid, function and how to optimize the most protective doses using biomarkers. In the future, this information will aid in the design of better HDAC inhibitors that could be used as even more effective drug treatments for neurological conditions.